PgmNr M281: CRISPR/Cas9 Genome Editing Pipeline for Mice and Rats.

Authors:
Thom Saunders; Wanda Filipiak; Galina Gavrilina; Anna LaForest; Corey Ziebell; Michael Zeidler; Elizabeth Hughes


Institutes
University of Michigan, Ann Arbor, MI.


Abstract:

CRISPR/Cas9 is a RNA guided nuclease that produces double strand breaks in DNA molecules and chromosomes. Mouse and rat zygotes with chromosome breaks may repair breaks with non-homologous endjoining (NHEJ) or homology directed repair (HDR) pathways. Repair NHEJ by often produces deletions and insertions in critical regions for gene expression that causes gene knockouts as a result. When the zygote copies new DNA sequence from a DNA donor template during HDR a new DNA sequence will be expressed from the targeted locus. Genes for the CRISPR/Cas9 pipeline are identified by investigators and the Transgenic Core designs sgRNA targets in critical regions with online algorithms. High scoring sgRNAs are cloned for in vitro sgRNA transcription. Activity of sgRNA is validated in two steps: 1) cleavage of PCR templates in vitro after mixture with Cas9 protein and 2) microinjection into mouse zygotes and testing blastocysts for NHEJ repair. Rat gene sgRNAs are tested by transfection of rat cells and identification of NHEJ mutations. Results will be presented on 1) producing gene knockouts in mouse and rat strains, 2) introducing coding SNPs knockins with oligonucleotide in mice and rats, 3) producing reporter gene knockins in mice and rats, 4) producing floxed genes with a novel one-cut strategy in mice, and 5) reporter gene knockins into the Gt(ROSA)26Sor locus. Analysis shows that mosaic founders occur frequently. Mutations observed in founders vary from deletion/insertion of a few nucleotides to the deletion of several hundred base pairs. These patterns are observed in gene knockouts in both mouse and rat models. The efficiency of CRISPR/Cas9 targeting was lower in inbred C57BL/6J mice than in mixed genetic backgrounds (C57BL/6 and SJL). NHEJ inhibitors such as SCR7 were ineffective in a series of reporter knockin experiments. Experiments are underway to determine if the high fidelity Cas9 system (Cas9HF1) is as efficient as the wild type Cas9 endonuclease. Gene targeting with CRISPR/Cas9 is highly efficient, we guarantee the production of mouse and rat gene model knockouts with reagents designed and cloned in our Core facility. The efficiency of oligonucleotide knockins is lower, the majority of SNPs succeed. ROSA26 knockins are also efficient. The introduction of complex alleles such as multi-reporter knockins (e.g. iCre-P2A-mCherry) are least efficient and cannot always be guaranteed. Compared to preceding technologies, CRISPR/Cas9 technology has significantly increased access to mouse and rat genomes for the generation of biomedical research models.